Empirical rheology and pasting properties of soft-textured durum wheat (Triticum

turgidum ssp. durum) and hard-textured common wheat (T. aestivum)

Enoch T. Quaysona, William Atwella, Craig F. Morrisb, Alessandra Marti a,c,*

a Department of Food Science and Nutrition, University of Minnesota, 1334 Eckles Ave,

St. Paul, MN, 55108, USA

b USDA-ARS Western Wheat Quality Laboratory, Washington State University, E-202 Food

Science & Human Nutrition Facility East, Pullman, WA 99164, USA

c Department of Food, Environmental and Nutritional Sciences, Universita’ degli Studi di

Milano, via G. Celoria 2, 20133, Milan, Italy

*Corresponding author: Phone +1 612 625 2768. Fax +1 612 625-5272

E-mail amarti@umn.edu; alessandra.marti@unimi.it

Keywords

Puroindolines; kernel hardness; gluten aggregation; dough rheology

1

Abstract 1

Puroindoline (PIN1) proteins are the molecular basis for wheat kernel texture classification and 2

affect flour milling performance. This study investigated the effect of PINs on empirical 3

rheology and pasting properties in T. turgidum ssp. durum and T. aestivum. Soft wheat (cv. 4

Alpowa), durum wheat (cv. Svevo) and their derivatives in which PINs were deleted (Hard 5

Alpowa) or expressed (cv. Soft Svevo). Presence of PINs affected flour particle size and 6

damaged starch. PINs increased the pasting temperature and breakdown viscosity, while the 7

effect on peak viscosity and setback were not consistent. Presence of PINs was negatively 8

associated with GlutoPeak gluten aggregation energy and farinograph dough stability, suggesting 9

a weakening of the gluten matrix. As regards dough extensibility, the role of PINs was evident 10

only in common wheat: 5DS distal end deletion increased the resistance to extension, without 11

affecting the dough extensibility. This study showed PINs to have different impact on pasting 12

and rheological properties of T. aestivum and T. turgidum ssp. durum flours. 13

1 List of abbreviation

AU, arbitrary unit; BE, Brabender equivalent; BU, Brabender unit; FU, farinograph unit; GPU,

GlutoPeak unit; LT30, Loss of Torque 30 s after maximum torque; PIN, puroindoline protein;

SKCS, Single Kernel Characterization System.

2

1. Introduction 14

Puroindolines (PINs) are wheat endosperm proteins associated with starch granules. They 15

are considered minor components due to their low level (about 0.1%) in wheat (Dubreil et al., 16

1998). Despite the low level of occurrence, PINs play a key role in determining the kernel 17

hardness of wheat (Morris, 2002; Bhave and Morris, 2008), which is defined as the force 18

required to crush the kernels. The expression of PINs is controlled by Puroindoline a (Pin a) and 19

Puroindoline b (Pin b) genes (Morris, 2002; Bhave and Morris, 2008) located on the distal end 20

of the short arm of chromosome 5D (5DS). Functional expression of both genes results in soft 21

kernel texture while the presence of only one functional gene or mutation in either of the genes 22

results in hard kernel texture. 23

Common wheat (Triticum aestivum L) endosperm texture ranges from soft to very hard, 24

while durum (T. turgidum ssp. durum) – which does not contain the 5D chromosome and 25

therefore no PIN genes - has harder kernel texture. Kernel texture has been an important index in 26

wheat commercialization, with hard kernel wheat attracting higher purchase value (Turnbull and 27

Rahman, 2002), due principally to the higher protein content compared to soft wheat (Pauly et 28

al., 2013a). The different kernel texture of the grains influences milling and end-use quality 29

characteristics that have been extensively reported in recent reviews on this topic (Pauly et al., 30

2013a, b). Soft wheat requires less energy to mill, has higher break flour yield, smaller flour 31

particle size and less damaged starch compared to hard wheat (Martin et al., 2007). The 32

comparatively higher proportion of intact starch granules in soft wheat flours – together with the 33

lower protein content - result in lower water absorption compared to hard wheat flour. Flours 34

from soft wheat are used in making pastries and cookies while flours from hard wheat are used 35

3

for bread and other leavened products. On the other hand, durum wheat is considered the best 36

raw material for producing pasta and cous-cous. 37

The probable effect of PINs on dough rheology and product end-use quality has elicited 38

considerable interest over the last decade. Most of the studies were carried out using 39

fractionation/reconstitution experiments. Addition of purified PINs at 0.1% level produced 40

opposite effects on dough strength and extensibility for flours with good or poor bread making 41

performance (Dubreil et al., 1998). In particular, addition of PINs to good and poor bread quality 42

flours increased and decreased dough strength and extensibility, respectively. Rouille et al. 43

(2005) reported that adding 0.2% PINs to bread flour resulted in increased crumb grain fineness 44

without affecting the bread specific volume, suggesting that PINs affect gas cell stabilization in 45

bread dough (Pauly et al., 2013b). Similarly, Pauly et al. (2013c) recently reported PINs as 46

exerting a softening effect when present above 0.07% in biscuit flour, highlighting how the level 47

of PINs is critical to product quality. Although these studies expanded our knowledge about the 48

role of PINs in dough and product characteristics, they present some limitations. First, PINs were 49

usually added at levels higher than those naturally present in flour. Second, PINs isolation could 50

have altered their functional properties, likely affecting protein interactions and, thus, dough 51

rheology. Finally, Triton X-114 – which is generally used to isolate PINs – is very difficult to 52

remove from protein samples. Thus, the presence of this detergent could impact the outcome of 53

experiments (Pauly et al., 2013b). 54

About a decade ago, some authors investigated the effects of PINs on bread quality using 55

transgenic lines in which PINs were over-expressed. Hogg et al. (2005) demonstrated that 56

transgenic over-expression of PINs in common wheat decreased loaf volume and crumb grain 57

scores. Cytological processes (homoeologous recombination) have also been used to transfer PIN 58

4

genes to durum wheat producing soft durum lines (Gazza et al., 2011; Morris et al., 2011). The 59

driving force behind producing soft-textured durum varieties is the potential increase in durum 60

wheat production and end-use product applications. In theory, a broader, more diverse range of 61

end-use for durum wheat should drive consumer demand and, hence, production (Morris et al., 62

2015). On the effect of durum kernel modification on product, pasta cooking quality was 63

unaffected by the kernel hardness, whereas bread from durum wheat exhibited an increase in loaf 64

volume associated with kernel softening (Gazza et al., 2011). 65

Similarly, back-cross seven (BC7) of common soft wheat cultivar (Alpowa) was used to 66

produce near-isogenic hard kernel lines lacking puroindolines (Morris and King, 2008). No 67

information on the rheological properties of these near-isogenic wheat lines with modified kernel 68

texture (hard-textured and soft-textured) is available. 69

This study investigated the effects of PINs expression or deletion on pasting properties, 70

gluten aggregation, dough mixing and extensibility of soft-textured durum and hard-textured 71

wheat. It will contribute to improve our understanding of the role of PINs in wheat quality and 72

utilization. 73

2. Materials and methods 74

2.1 Wheat samples 75

Wheat cultivars (cvs.) Alpowa (soft wheat, T. aestivum L.), hard kernel Alpowa (Hard 76

Alpowa), durum wheat (T. turgidum L. ssp. durum) cv. Svevo, and soft kernel durum wheat cv. 77

Soft Svevo were used in the study. Hard Alpowa is a back-cross seven near-isogenic line of the 78

soft wheat cv. Alpowa that lacks the distal portion of short arm of chromosome 5D (Morris and 79

King, 2008). It involved crossing donor parents possessing Pin a and Pin b halotype genes with 80

white soft spring cv. Alpowa. F1 and F2 seeds were harvested, planted and allowed to self. F3 81

5

seeds from individual F2 plants were subjected to progeny phenotypic screening. A homozygous 82

hard plant was selected for backcrossing using Alpowa as recurrent male parent. The process 83

was repeated to identify plants homozygous for hardness trait (Hard Alpowa). Soft Svevo was 84

developed from recurrent back-crossing durum wheat cv. Svevo with Langdon durum that had 85

Pin a and Pin b which were translocated from chromosome 5D of soft wheat cv. Chinese Spring 86

(Morris et al., 2011). Alpowa and Hard Alpowa were grown in St. Paul (MN, US) and harvested 87

in 2014. Svevo and Soft Svevo were grown in Pullman (WA, US) in 2013. 88

Wheat grains were conditioned (14.5 g/100 g moisture for Alpowa and Soft Svevo; 15.5 89

g/100g for Hard Alpowa; 16.5 g/100 g moisture for Svevo) and subsequently milled with a 90

Quadrumat Junior (C.W. Brabender Inc., South Hackensack, NJ, USA) according to Approved 91

Method 26-50.01 (AACCI, 1999). 92

2.2 Single Kernel Characterization System 93

Single Kernel Characterization System (SKCS) hardness values of the wheat cultivars 94

were determined according to Approved Method 55-31.01 (AACCI, 1999). 95

2.3 Physicochemical characterization of flours 96

Moisture content was measured by drying the sample at 180 °C for 4 min in an infrared 97

balance (MB 45, OHAUS, Parsippany, NJ). Damaged starch levels were measured according to 98

Approved Methods 76-31.01 (AACCI, 1999). Flour particle size distribution was analyzed 99

according to the Approved Method 55-60.01 (AACCI, 1999). 100

2.4 Pasting Properties 101

The pasting properties of the wheat flours were determined using a Micro-Visco 102

Amylograph device (C. W. Brabender Instruments, South Hackensack, NJ). Fifteen grams of 103

6

flour (14% moisture basis) were dispersed in 100 mL distilled water and stirred at 250 rpm. The 104

following temperature profile was applied: mixing at 30°C for 3 min, heating from 30 °C to 95 105

°C at a rate of 7.5 °C/min, holding at 95 °C for 5 min, cooling from 95 °C to 30 °C at a rate of 106

-7.5 °C/min, and holding at 30°C for 2 min. The following indices were considered: (i) Pasting 107

temperature (temperature at which an initial increase in viscosity occurs); (ii) Peak viscosity 108

(maximum viscosity achieved during the heating cycle); (iii) Peak temperature (temperature at 109

the maximum viscosity); (iv) Breakdown viscosity (index of viscosity decrease during the 110

holding period, corresponding to viscosity difference between peak and after holding at 95 °C); 111

(v) Setback viscosity (index of the viscosity increase during, corresponding to the difference 112

between the final viscosity at 30 °C and the viscosity reached after the holding period at 95 °C). 113

Peak viscosity, breakdown, and setback viscosities were expressed in Brabender Units (BU). 114

Pasting temperature and peak temperature were expressed in °C. For each sample the test was 115

run in triplicate. 116

2.5 GlutoPeak Test 117

Gluten aggregation properties of flour samples were evaluated using the GlutoPeak 118

device (C.W. Brabender Inc., South Hackensack, NJ, USA), as reported by Chandi and 119

Seetharaman (2012). An aliquot of 8.5 g of flour (14% moisture basis) was dispersed in 9.5 g of 120

0.5M CaCl2. Sample temperature was maintained at 34 °C by circulating water through the 121

jacketed sample cup. The paddle was set to rotate at 1,900 rpm and the test was carried out for 7 122

minutes. The main indices automatically evaluated by the software provided with the instrument 123

(GlutoPeak v. 2.1.0) were: (i) Peak maximum time (expressed in seconds), corresponding to the 124

time before torque decreased due to gluten break down; (ii) Maximum torque (expressed in 125

Brabender Equivalents - BE), corresponding to the peak occurring as gluten aggregates; (iii) 126

7

Energy to peak (expressed in GlutoPeak Unit - GPU), corresponding to the area under the curve 127

until the maximum torque. In addition, the loss of torque 30 s after maximum torque (%) - 128

corresponding to the decrease in torque 30 s after peak (LT30s) – was calculated. For each 129

sample the test was run in triplicate. 130

2.6 Mixing Properties 131

The behavior of the dough during mixing was measured using a Farinograph - AT (C.W. 132

Brabender Inc., South Hackensack, NJ, USA) equipped with a 50 g mixing bowl and according 133

to Approved Method AACCI 54 -21.02 (AACCI 2000). The following indices were considered: 134

(i) Water absorption (expressed in per cent), corresponding to the amount of water needed to 135

reach the optimal consistency (500±20 Farinograph Unit, FU); (ii) Dough development time, 136

corresponding to the time from first addition of water to the point of maximum consistency 137

range; (iii) Stability, corresponding to the time difference between when the curve reaches 138

(arrival time) and leaves (departure time) the 500 FU line. Each dough sample was analyzed in 139

duplicate. 140

2.7 Dough Extensibility 141

Dough extensibility was measured with a micro-Extensograph instrument (C.W. 142

Brabender Inc., South Hackensack, NJ, USA) on a 20 g dough piece, according to the 143

manufacturer’s manual. Dough was prepared according to AACCI Approved Method 54-10.01 144

in the 50 g test bowl of the farinograph, with addition of 2% NaCl, on a flour weight basis. The 145

following parameters were considered: (i) Resistance to extension (expressed in BU), measured 146

50 mm after the curve has started and is related to the elastic properties of dough; (ii) Maximal 147

resistance to extension (expressed in BU); (iii) Extensibility (expressed in mm) corresponding to 148

8

distance at sample rupture; (iv) Energy (expressed in arbitrary units, AU) corresponding to the 149

area under the curve; (v) Ratio, corresponding to the ratio between extensibility and resistance; 150

(vi) Ratio Max, corresponding to the ratio between extensibility and maximal resistance to 151

extension. Measurements for each sample were performed in duplicate and from each dough two 152

subsamples were tested. 153

2.8 Statistical analysis 154

Analysis of variance (ANOVA) was performed utilizing Statgraphics XV version 15.1.02 155

(StatPoint Inc., Warrenton, VA, USA). Puroindolines presence was used as a factor. When the 156

factor effect was found to be significant (p≤0.05), significant differences among the respective 157

means were determined using Fisher’s Least Significant Difference (LSD) test. 158

3. Results and Discussion 159

3.1 Kernel and flour characterization 160

Physical characteristics of wheat samples are summarized in Table 1. Durum wheat 161

Svevo and Hard Alpowa samples exhibited higher SKCS hardness values than Soft Svevo and 162

Alpowa soft wheat, respectively. Kernel texture in wheat is controlled by Pin a and Pin b genes: 163

soft wheat has both functional Pin a and Pin b, while hard wheat has either one or a mutation of 164

either Pin a or Pin b. Durum wheat does not contain any of these endosperm-softening PIN 165

genes, and therefore, it has very hard kernels. Similarly, the Hard Alpowa is missing the distal 166

portion of chromosome 5DS and thus is also missing the PIN genes. The differences in PIN 167

expression affected the flour protein concentration. Flours from grains without PINs (Svevo and 168

Hard Alpowa) showed higher protein content than the corresponding samples with PINs (Table 169

1). The effect of PINs expression on protein content needs further investigation. 170

9

Kernel hardness affects various flour properties including particle size distribution and 171

damaged starch (Table 1). As regards particle size, milling Svevo grain (using a mill for common 172

wheat) resulted in two main fractions: one fraction with particle size ≥75 µm (55% of total) and 173

another with particle size <75 µm (45% of total). PIN expression and the consequential soft 174

kernel texture affected milling properties of the sample. Indeed, flour from Soft Svevo had a 175

higher percentage of particles < 75 µm (75% of total) and lower percentage of ≥ 75 µm (25% of 176

total). Moreover, differences in particle size contributed to differences in color between the two 177

flours which is in agreement with Gazza et al. (2011). Color attributes - with particular regards to 178

yellowness - are of great importance in durum wheat quality evaluation. Svevo exhibited a 179

higher yellowness (b*) than Soft Svevo (20.0 vs 14.4, Fig. S1). Differences in color could also 180

be attributed to differences in damaged starch granules, which do not reflect light as effectively 181

as intact granules (Miskelly, 1984). 182

The deletion of the chromosome 5DS distal end where the Pin a and Pin b genes are 183

located in Hard Alpowa resulted in an increase in kernel hardness and consequently larger flour 184

particle size with a higher percentage of particles ≥ 75 µm compared to Alpowa (65 vs 48% of 185

total for Hard Alpowa and Alpowa, respectively). 186

Differences in kernel texture also affected the level of damaged starch in the flours. As 187

expected, in Alpowa and Soft Svevo, the percentage of damaged starch was significantly 188

(p0.05) lower than in Hard Alpowa and Svevo, respectively (Table 1). The level of damaged 189

starch in the flour contributes to the water absorption capacity of the flour during mixing. 190

Damaged starch absorbs about twice its own weight of water, which is about 5 times greater than 191

that of intact starch (Stauffer 2007), and depending on its level, makes a significant contribution 192

to the overall water absorption capacity of flours (Cauvain, 2009). 193

10

3.2 Pasting properties 194

The effects of PINs on flour pasting properties are shown in Fig. 1. Soft Svevo showed 195

higher pasting temperature, peak viscosity, breakdown viscosity and setback viscosity values 196

than Svevo (Fig. 1Aand Table 2). The significantly (p0.05) higher pasting temperature in Soft 197

Svevo compared to Svevo may be attributed to the presence of PINs, in agreement with a 198

previous study on starch isolated from transgenic rice (Wada et al., 2010). PINs – which are 199

localized at the starch surface (Feiz et al., 2009) - could inhibit the access of water to starch, 200

which in turn would result in an extended time (and higher temperature) for starch to gelatinize 201

and to reach peak viscosity. Interestingly, the detail in Fig. 1A showed for Soft Svevo a delay in 202

granule swelling (related to increased viscosity) compared to Svevo, likely suggesting an effect 203

of PINs on starch swelling at temperatures below 85 °C. As the temperature increased, Soft 204

Svevo showed a higher peak viscosity than Svevo, indicating greater swelling capacity. 205

However, Soft Svevo exhibited a slightly slower gelatinization rate, since it reached the peak 206

viscosity at around 10 min, whereas Svevo reached the maximum value about 30 seconds earlier. 207

PINs therefore seem to tolerate temperature, moderating temperature effect on starch properties. 208

During the holding time at 95 °C for 5 min, Soft Svevo showed higher stability to high 209

temperature and mixing as indicated by the lower breakdown value compared to Svevo. Finally, 210

during the cooling step, starch in Soft Svevo showed a greater ability to reassociate in a new 211

structure that exhibited higher viscosity compared to Svevo, therefore suggesting a higher 212

retrogradation tendency. 213

As regards T. aestivum, Hard Alpowa showed a significant (p0.05) decrease in pasting 214

temperature and breakdown viscosity than Alpowa (Fig. 1B and Table 2). These results are 215

consistent with the results obtained for Svevo and Soft Svevo, which could be related to the 216

11

impact of PINs expression (Svevo vs Soft Svevo) or 5DS distal end deletion (Alpowa vs Hard 217

Alpowa) on pasting temperature and paste stability during the holding time at 95 °C. As regards 218

the impact of PINs on viscosity during heating and cooling, Hard Alpowa showed higher peak 219

viscosity and setback values than Alpowa. These results are in agreement with those obtained 220

from reconstitution studies on common wheat flours which suggested that PINs affect pasting 221

profiles by restricting starch water absorption and swelling in a diluted system, as in the case of 222

the Micro-ViscoAmlograph test (Pauly et al., 2012; Debet and Gidley, 2006). Conversely, the 223

impact of 5DS distal end deletion on peak viscosity and setback is not consistent with the trend 224

observed for PINs expression (Fig. 1A). 225

Overall, the results on pasting properties suggest that PINs impact the temperature for 226

onset of gelatinization (pasting temperature) and also the breakdown viscosity. However, the 227

effect of PINs on starch swelling (peak viscosity) and retrogradation tendency (setback) remains 228

unclear since it is apparently dependent on the type of wheat (i.e. T. aestivum or T. turgidum ssp. 229

durum). Decreases in viscosity during heating and cooling have also been associated with an 230

increase in damaged starch (Liu et al., 2014; Leon et al., 2006). This is consistent with our data 231

on Svevo and Soft Svevo. On the contrary, since Hard Alpowa contained higher levels of 232

damaged starch than Alpowa (Table 1), a lower maximum viscosity would have been expected 233

for Hard Alpowa compared to Alpowa. This leads to the conclusion that PINS likely do affect 234

flour pasting profiles. 235

3.3 Gluten Aggregation Properties 236

Fig. 2 presents the impact of PINs on gluten aggregation profile obtained by the 237

GlutoPeak test. The parameters associated with the aggregation curves are reported in Table 2. 238

During the test, the sample slurry is subjected to intense mechanical action, promoted by the 239

12

speed (1,900 rpm) of the rotating element, which facilitates the formation of gluten, and a rapid 240

increase of the torque curve is registered until the maximum torque is reached. Further mixing 241

depolymerizes the network, with a concomitant decline in torque. The loss of torque 30s (LT30s) 242

after maximum torque is an index of gluten strength during prolonged mixing. 243

In Svevo, PINs expression caused a significant (p0.05) decrease in maximum torque 244

with no effect on the peak maximum time (Fig. 2A; Table 1). Consequently, GlutoPeak test 245

energy, which is the area under the mixing curve to peak and takes into consideration the 246

maximum torque and peak maximum time indices, decreased when PINs were expressed. This 247

energy has been shown to correlate with gluten strength (measured as gluten index) and pasta 248

cooking quality (Marti et al., 2014). Finally, 30s after maximum torque, Soft Svevo showed a 249

significantly (p0.05) higher LT30s value than Svevo indicating a higher loss of torque and thus 250

greater gluten breakdown due to over-mixing compared to Svevo. 251

The 5DS distal end deletion caused a significant (p0.05) decrease in peak maximum 252

time and an increase in maximum torque and energy that suggest the presence of stronger gluten 253

in Hard Alpowa compared to Alpowa (Fig. 2B), as supported by the energy value (Table 2). 254

Among the GlutoPeak indices, the energy value is considered the most significant parameter for 255

the prediction of the conventional parameters related to dough mixing such as stability, 256

extensibility, and tenacity (Marti et al., 2015). 257

Since both PINs expression and 5DS distal end deletion did not affect the glutenin and 258

gliadin genes, and therefore the gluten composition of the samples (data not shown), differences 259

in gluten aggregation kinetics among the samples were likely related to PIN proteins. In flour, 260

PINs are present at the starch granule surface and associate with polar lipids (Feiz et al., 2009). 261

During dough mixing, they are removed from the granule surface and become incorporated in 262

13

the gluten network, together with polar lipids (Finnie et al., 2010; Pauly et al., 2012). It may be 263

hypothesized that PIN-polar lipid complexes interact with gluten proteins and delay and limit the 264

extent of gluten aggregation. 265

3.4 Mixing Properties 266

The farinograph profiles of wheat samples are reported in Fig. 3. Soft Svevo showed 267

lower water absorption capacity than Svevo, and similarly Alpowa showed lower water 268

absorption capacity than Hard Alpowa (Table 2), reflecting the effect of high starch damage of 269

the milling products from hard kernels compared with soft kernels (Table 1). Moreover as a 270

consequence of PINs expression (Soft Svevo vs Svevo) dough development time and stability 271

decreased (Fig. 3A). Indeed, differences in protein content between particular samples might 272

account for the differences in dough strength. The protein contents of the samples of PINs 273

expression and deletion (Table 1) are in agreement with previous reports that showed a decrease 274

in flour protein when PINs were transgenically expressed (Hogg et al., 2005). Since in the 275

present study each set of samples was grown under the same environmental conditions, results 276

suggest that differences in protein content were solely related to presence of PINs that affected 277

the mixing properties of the dough. 278

Our findings on mixing properties are in agreement with those reported by Hogg et al. 279

(2005). On the contrary, studying the effects of grain texture on pasta-making and bread-making, 280

Gazza et al. (2011) found that soft durum lines had higher stability than hard durum lines (cv. 281

Langdon), likely due to the inability of damaged starch in hard durum lines flour to hold all the 282

water absorbed initially. Moreover, soft-textured durum wheat lines did not differ from the hard 283

durum lines in terms of dough mixing time (Gazza et al., 2011). These contrasting results 284

confirm the observation made by Pauly et al. (2013c) that when puroindolines were added to 285

14

biscuit flour at levels higher than 0.07%, it affected the dough texture. This suggests that for 286

PINs to affect flour-dough quality parameters such as mixing time and stability, they will have to 287

be present at a certain threshold level. 288

The 5DS distal end deletion (Alpowa vs. Hard Alpowa) increased both dough 289

development time and stability (Table 2; Fig. 3B). The absence of PINs likely improved gluten 290

protein interaction, resulting in increased dough development time and stability. The results 291

agree with the typical farinograph profiles of strong and weak dough wheat flours. Usually, 292

strong dough flours require higher amounts of water and longer mixing times to form a fully 293

developed gluten network, which exhibits longer stability than flours with poor bread-making 294

performance (Cauvain, 2009). Some of the differences in farinograph measurements (e.g. water 295

absorption) could be attributed to protein content, damaged starch and flour particle size, 296

whereas the differences in dough development time and stability are generally attributed to 297

different types of gluten (Matsuo and Irvine, 1970). 298

3.5 Dough Extensibility 299

The tensile properties of dough were carried out on a “micro scale” using 20 g of dough 300

and a micro-Extensograph which records the resistance of dough to stretching and the distance 301

the stretched dough covers before breaking. The resistance of dough to the deformation forces is 302

expressed as energy value and correlates well with the gas retention capacity of dough, volume 303

of the end product after baking, handling properties, and is also taken as a guideline parameter 304

for flour blending operations at milling facilities (Ktenioudaki et al., 2010). Hard wheat flours 305

generally show high extensibility and a relatively high resistance to extension, a good balance of 306

which is essential to hold gas bubbles during the fermentation of bread dough and other leavened 307

15

products. On the other hand, doughs from soft wheat flours show high extensibility but low 308

resistance to extension which makes them suitable for pastries and cakes. 309

Dough strength and extensibility of Soft Svevo were similar to Svevo (Table 2). Indeed, 310

the PIN-possessing chromosome translocation does not alter any of the gluten proteins from the 311

parent durum variety (Morris et al., 2011; Morris and King, 2008). For both Svevo and Soft 312

Svevo, dough extensibility did not change for the different resting times (45, 90 and 135 min, 313

data not shown). Gluten network in T. turgidum ssp.durum seems to be too tenacious for PINs to 314

have a noticeable effect on dough extensibility. The results of the present work partially agree 315

with previous studies. Gazza et al. (2011) reported no differences in dough extensibility 316

(measured as Alveograph L value) between durum wheat with PINs and one with no PINs. On 317

the other hand, dough strength (Alveograph W value, which is energy required to blow and break 318

a bubble of dough) was significantly lower in soft durum lines compared with hard durum lines 319

(Gazza et al., 2011). The Alveograph, however, is performed using a constant level of water 320

absorption such that dough rheology is confounded with flour water absorption. Performing 321

reconstitution experiments, Dubreil at al. (1998) showed that addition 0.1% of PINs drastically 322

decreased the dough strength (Alveograph W) and increased the extensibility (measured as 323

Alveograph L) in wheat flours with poor and medium bread-making performances. On the 324

contrary, when PINs were added to a flour of good bread-making quality, an increase in W and a 325

decrease in L were observed. Moreover, regardless of the bread baking quality of flour, tenacity 326

(measured as Alveograph P) increased in the presence of PINs. It is important to keep in mind 327

that contrasting results could be related to differences between the techniques. Firstly, the 328

Extensograph stretches the dough in uniaxial mode while Alveograph expands the dough in all 329

directions. Secondly, Extensograph works with doughs prepared to optimum hydration levels 330

16

suited for different processing applications as in the real industrial world, whereas a constant 331

amount of hydration is used in an Alveograph. 332

5DS distal end deletion did not affect dough extensibility (Table 2). On the other hand, 333

Hard Alpowa showed a significantly (p0.05) higher resistance to extension and strength than 334

Alpowa, suggesting that the presence of PINs improved the resistance to extension only in the 335

case of weak flours. In addition, Hard Alpowa exhibited higher ratio values than Alpowa (Table 336

2). Since ratio indices are a measure of the balance between elasticity and extensibility, high 337

values are generally indicative of tenacious/strong dough. 338

4. Conclusions 339

The study of the role of PINs on physical properties of doughs prepared from T. aestivum 340

and T. turgidum ssp. durum - in which the genes for PINs were deleted or expressed, respectively 341

– highlighted the following points: (i) wheat samples with PINs exhibited delayed starch 342

gelatinization and less capacity to maintain the granule integrity at high temperature, (ii) wheat 343

samples with PINs exhibited delayed gluten aggregation, likely due to the formation of PIN-lipid 344

complexes that surround gluten proteins, (iii) wheat samples with PINs exhibited decreased 345

dough stability, an indication that PINs interact with gluten, and (iv) the impact of PINs on starch 346

swelling and dough extensibility is species- or variety-dependent. 347

The effects of PINs on dough rheological properties should be confirmed by investigating 348

a larger number of varieties. Further studies should investigate the nature of PIN-gluten 349

interactions and their potential role in product quality. 350

Acknowledgments 351

17

The authors kindly acknowledge Dr. J. Anderson (University of Minnesota) for growing 352

Alpowa and Hard Alpowa, Professor Maria Ambrogina Pagani (University of Milan) for critical 353

reading of the manuscript, and CW Brabender (South Hackensack, NJ, USA) for the use of their 354

analytical equipment. The immense contribution and inspiration from Dr. Koushik Seetharaman 355

is also recognized. 356

References 357

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1

Table 1. Kernel hardness, and flour particle size and damaged starch content of wheat samples

possessing or lacking PINs

Svevo Soft Svevo Alpowa Hard Alpowa

SKCS 73 17 16 98

Particle size (%)

<75μm 44.1±1.3 73.9±0.9 51.5±1.2 34.8±1.5

≥75μm 54.1±1.5 23.9±1.2 47.9±0.9 63.5±1.5

Protein g/100g 15.9±0.23 14.8±0.21 12.3±0.22 14.8±0.10

Damaged Starch (g/100gdb) 12.1d 4.8b 4.5a 10.9c

SKCS - single kernel characterization system. Values in the same row with the same letters are

not significantly different (p0.05)

2

Table 2. Gluten aggregation and dough mixing and extensibility properties of wheat samples

possessing or lacking PINs

Values in the same row with the same letters for each test are not significantly different (p0.05)

Svevo Soft Svevo Alpowa Hard Alpowa

Micro-Visco

Amylograph

test

Pasting temperature (C) 58.3a 60.8b 60.8b 59.0a

Peak viscosity (BU) 723c 849d 566a 638b

Breakdown viscosity (BU) 123a 167b 323d 303c

Setback viscosity (BU) 799c 988d 631a 746b

GlutoPeak

test

Peak maximum time (s) 57.7b 58.3b 132.0c 50.3a

Maximum torque (BE) 52c 34b 30a 54c

Energy to peak (GPU) 2166c 762a 765a 1576b

LT 30s (%) 18a 20b 30d 27c

Farinograph

test

Water absorption (%) 76.6 60.6 57.2 68.8

Development time (min:s) 04:51 01:50 01:35 05:31

Stability (min:s) 03:20 02:35 02:13 08:30

Micro-

Extensograph

test

Extensibility (mm) 43a 43a 71b 72b

Resistance (BU) 100b 95b 77a 101b

Maximum resistance (BU) 101b 96ab 82a 108b

Ratio 2.3a 2.2a 1.1b 1.4c

Ratio Max 2.4a 2.2a 1.2b 1.5c

Energy (AU) 3589a 3409a 4710b 6286c

3

Figure Captions

Figure S1. Pictures showing colors of flours (a) Svevo and (b) Soft Svevo.

Figure 1. Pasting profile of (a) Svevo (black line) and Soft Svevo (grey line) flours and (b)

Alpowa (grey line) and Hard Alpowa (black line). Dotted lines represent sample temperature.

Figure 2. Gluten aggregation profile of (a) Svevo (black line) and Soft Svevo (grey line) flours

and (b) Alpowa (grey line) and Hard Alpowa (black line).

Figure 3. Mixing profile of (a) Svevo (black line) and Soft Svevo (grey line), (b) Alpowa (grey

line) and Hard Alpowa (black line).

4

Fig. S1. Pictures showing colors of flours (a) Svevo and (b) Soft Svevo.

A

B

5

Figure 1. Pasting profile of (a) Svevo (black line) and Soft Svevo (grey line) flours and (b)

Alpowa (grey line) and Hard Alpowa (black line). Dotted lines represent sample temperature.

6

Figure 2. Gluten aggregation profile of (a) Svevo (black line) and Soft Svevo (grey line) flours

and (b) Alpowa (grey line) and Hard Alpowa (black line).

7

Figure 3. Mixing profile of (a) Svevo (black line) and Soft Svevo (grey line), (b) Alpowa (grey

line) and Hard Alpowa (black line).